Infections due to Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, are the leading causes of morbidity and mortality in both HIV infected and immune competent people. A major factor contributing to virulence of Mtb is its intrinsic slow growth rate and the ability to maintain a non-replicative persistent (NRP) state. Mtb employs several two-component regulatory signal transduction systems (2CRS) for its optimal survival upon infection, and the MtrAB is the only essential 2CRS. DNA replication is essential for multiplication of organisms and is believed to be regulated at the initiation step. The genetic elements responsible for the regulation of Mtb replication and or the factors that promote NRP state in vivo are largely unknown. DnaA protein initiates this process at a unique site on the genome called oriC. Our recent data indicate that the MtrA response regulator (RR) binds to oriC and the dnaA promoter. Our proposal focuses on the regulation of initiation of DnaA mediated oriC replication by MtrA RR in synchronously replicating cells. The novel Mtb dnaA cold sensitive mutants that we have generated will be used to obtain synchronously replicating population. We hypothesize that Mtb DnaA mediated oriC replication is regulated in part by the MtrA RR in a phosphorylation dependent manner. A consequence of this regulation will determine if, upon infection, DNA replication will continue resulting in bacterial multiplication or will stall/ shut-down promoting metabolically active, but NRP state. We will evaluate the contribution of the MtrAB signal transduction pathway on oriC replication by biochemical, genetic and physiological approaches. In the Specific Aim 1 we will evaluate MtrA binding to oriC both in the absence and presence of DnaA, and the consequence of these interactions on the oriC replication. In the Specific Aim 2, we will construct and characterize constitutively active MtrA~P (MtrA~P*) in an effort to evaluate the role of MtrA in Mtb proliferation. In the Specific Aim 3, we will evaluate the MtrA occupancy of oriC under synchronous replication conditions in cells producing MtrA~P*. Our proposed experiments will define how the DnaA mediated oriC replication is regulated by the MtrAB system. It is hoped that these experiments will define how the DNA replication process in Mtb is regulated during active replicative and the NRP growth states, hence will improve our ability to control Mtb infections. PUBLIC HEALTH RELEVANCE: Tuberculosis remains a major public health crisis despite being a curable disease. Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is the leading cause of morbidity and mortality in both HIV infected and immune competent people. The characteristic features of Mtb are its slow growth and the ability to maintain a non-replicative persistent state. Mtb employees several two-component regulatory systems (2CRS) of signal transduction for its optimal survival in vivo and the MtrAB system is the only essential 2CRS. DNA replication is essential for cell duplication and subsequent proliferation. Our research proposal focuses on understanding how DNA replication, one of the important factors contributing to persistence, is regulated upon infection by the MtrAB system. This knowledge will ultimately improve our ability to control Mtb infections.